Vibrio cholerae are aquatic bacteria that cause the severe diarrheal disease cholera when ingested by humans in contaminated water. Cholera afflicts an estimated 5 million people annually, causing over 100,000 deaths. Currently the predominant animal models for V. cholerae are infant mice and rabbits. However, these models are expensive, difficult, unpleasant, and do not reproduce true disease states, as they are not natural hosts for V. cholerae. V. cholerae naturally inhabit several aquatic niches, including associations with shellfish, insect egg masses, and plankton. Recently V. cholerae were also found to colonize the intestinal tracts of vertebrate fish. Our central hypothesis is that the zebrafish, Danio rerio, is an ideal natural animal host model for V. cholerae that will make new studies of both pathogenesis and environmental colonization possible. Zebrafish have been utilized extensively in the past 20 years as model organisms for many different biological processes, including infectious disease models. Preliminary data indicate that V. cholerae colonize the zebrafish intestine to very high levels when administered either directly into the digestive tract via gavage or when simply added to water in which zebrafish are incubated. The latter is a much more natural, representative model for colonization than any existing animal model, and we are likely to learn important new information about the colonization process by utilizing zebrafish as an animal model. In addition to colonizing the zebrafish intestine, V. cholerae that have a functional virulence regulatory system also kill zebrafish through an unknown mechanism. Therefore, zebrafish may also be a good model for studying V. cholerae disease processes. The objective of this proposal is to establish zebrafish as a functional natural animal model for V. cholerae. We will determine the sites of V. cholerae colonization of zebrafish. We will establish a timeline for colonization, such that future experiments can utilize the most relevant time points for collecting data. We will determine the infectious dose required for V. cholerae colonization of zebrafish, which is important for the design of future experiments and will illuminate the dynamics of free swimming V. cholerae and fish in the natural environment. Finally, we will determine how secreted factors from V. cholerae cause fish death. Because the major virulence regulon is required for causing zebrafish death, V. cholerae virulence genes must be induced in the presence of zebrafish. Our long term goal is to identify the signals that induce virulence during human infection such that virulence can be disrupted therapeutically. The zebrafish model presents an entirely new opportunity for identifying these signals. Establishment of zebrafish as a natural animal host model should prove extremely useful to V. cholerae researchers studying not only colonization and disease processes but also the environmental lifestyle of V. cholerae, which is very important for understanding the V. cholerae reservoirs that can produce epidemic or pandemic cholera outbreaks. Therefore establishment of this new model should have a broad impact on V. cholerae research worldwide.